Interleukin-21 rs2055979 and Interleukin-21 receptor rs3093390 genetic variants and hepatitis C virus chronic infection.

Aim
The aim of this case-control study was to investigate association of G/T IL-21 (rs2055979) and C/T IL-21R (rs3093390) gene polymorphisms with chronic hepatitis C virus in Iranian Patients.


Background
Interleukin 21 (IL-21) has a significant function in the regulation of cellular immune responses. Its exclusive receptor, IL-21R, expressed on the surface of T, B and NK cells and is important for the proliferation and differentiation of these immune cells. Hence, it was suggested to be involved in response to viral infections.


Methods
This study follows a case-control study design and blood samples were collected from 290 patients with chronic HCV and 290 controls for both genes. Genomic DNA was extracted and then for each position, SNP was genotyped by the dedicated PCR and restriction fragment length polymorphism (RFLP) method. The results were analyzed by SPSS software using logistic regression and Chi-square tests.


Results
Genotype frequencies of GG, GT and TT in IL21 gene (rs3093390) were found to be 27.6%, 48.3%, 24.1% and 25.2%, 55,5%, 19,3% respectively in HCV infected patients and control group. For IL21R gene (rs2055979) genotype frequencies of CC, CT and TT were 63.8%, 31.4%, 4.8% and 61.4%, 29.7%, 9.0% respectively in HCV infected patients and control group. P values for genotype and allele frequencies were p=0.188, p=0.769 for IL21 gene, and p=0.144, p=0.179 for IL21R gene respectively.


Conclusion
As a result, there is no evidence for an association between IL-21 (rs2055979) and IL-21R (rs3093390) gene polymorphisms with chronic hepatitis C virus in Iranian Patients.

Introduction 1 Hepatitis C virus infection has become global health concern. More than 184 million people have been infected with hepatitis C virus (HCV) in the world (1, The vigor of the immune response plays an important role in the outcome of viral infections (6). Cytokines as key components of immune system are intercellular mediators involved in viral control and damages being induced by infection (7). Interleukin-21 (IL-21) is the most recently discovered member of type-I cytokine superfamily that has regulatory effects on the immune system. Interleukin 21, mainly produced by a range of differentiated CD4+ T-cell subsets (8). IL-21 specifically sustains CD8+ T cell effector activity and prepares a mechanism of CD4+ T cell helper during chronic viral infections (9). It also has a significant influence on the regulation of B-cell functions. It promotes the differentiation of antigen-stimulated B cells into memory and antibody-secreting plasma cells, affects IgE production, and induces Ig switching to IgG1 and IgG3 production (10). Also, several in vivo studies in animal models have demonstrated that IL-21 is vital for controlling chronic viral infections (11,12). On the other hand, its private receptor, IL-21R, shares the common γ-chain with IL-2R, IL-4R, IL-7R, IL-9R, and IL-15R. Engagement of IL-21 to its cognate receptor activates Jak1, Jak3 and subsequently STATs notably STAT1, STAT5a, STAT5b and predominantly STATE. Besides, IL-21 signals through the canonical PI3K and MAPK pathways (13). As multiple immune cell types express the IL21R, this cytokine exerts pleiotropic modulatory functions in both innate and adaptive cells including NK cells, B cells, CD4 and CD8 T cells (13,14). Among the host factors, single nucleotide polymorphisms (SNPs) in immune genes such as interleukins can play significant roles in regulation the host immune response to the viral infections (15). In addition, some studies demonstrated that single nucleotide polymorphisms in cytokines and their receptor genes may contribute to the susceptibility of disorders and diseases (11,(16)(17)(18)(19)(20)(21)(22). Recently, many studies supported that variant SNPs located within the IL-21 gene have genetic association with susceptibility to different diseases particularly autoimmune diseases (18,23,24). Herein, in the present study, association of intronic polymorphisms in IL-21 gene (rs2055979) and its dedicated receptor (rs3093390) with chronic HCV infection was distinguished.

Subjects
In this Case-control study, for each gene, we analyzed a total of 580 subjects, including 290 chronic HCV patients who admitted in Gastroenterology and Liver Diseases Research Center, Shahid Beheshti University of Medical Sciences, and also 290 healthy individuals as a control group.

Serological Assays
Control group were healthy individuals without any liver diseases along with negative results for anti-HCV antibody tested by ELISA and then the presence or absence of viral particles in serum was determined by reverse transcription-PCR (RT-PCR). Selection criteria for patients group were positive results for anti-HCV antibody ELISA. RT-PCR and HCV RNA PCR were tested for anti HCV antibody positive cases.

Genomic DNA extraction and genotyping
Total Genomic DNA was extracted from EDTAanticoagulated peripheral blood samples of both patients and control group by the standard salting-out method (25). For each studied gene, polymorphic region was amplified by the polymerase chain reaction (PCR) in a total volume of 25 μL, using PCR reagents and specific primers. The annealing temperature (Tm) for the PCR was 62°C for IL21 and 61.2°C for IL21R gene. Then the PCR products were analyzed by incubation in a 37° centigrade water bath, the RFLP products were analyzed by 3% agarose (Roche, Germany) gel. Additionally, to confirm the PCR-RFLP assay, in each position, about 10% of total samples with different kinds of genotypes were selected and then their PCR products were sequenced. The PCR-RFLP products size, restriction site of enzymes and genotypes, for each SNP are shown in Table 1.

Statistical analyses
Genotype and allele frequencies of IL-21 and IL-21R were compared between HCV infected cases and controls using the Chi-square (χ 2 ) test. To adjust for confounding factors including age and gender, logistic regression analysis was used. To estimate the association between individual polymorphisms and chronic HCV, we were used odds ratios (OR) and 95% Confidence Intervals (95% CI). P-values of less than 0.05 were considered statistically significant. All statistical analyses were performed using the SPSS software (version 22; SPSS, Chicago, IL, USA).

Results
In this study, the case group consisted of 290 chronic HCV patients and the control group consisted of 290 healthy individuals. The number of males and females participated in this study is shown in About 10% of total samples with different kinds of genotypes were selected and then their PCR products were rechecked by the sequencing method and were concordant with the original RFLP results. Result of sequencing for heterozygous genotypes are shown in Figure 3.   The genotype and allelic frequencies of IL-21 and IL-21 receptor genes variation of the patient and control groups are presented in Table 4. The chi-square test was used to analyze the association between the genotypes and hepatitis status, the result is also shown in Table 4.
According to result of the Chi-square test, there were no statistically significance in the distribution of genotypes and allele frequencies between the patients and control groups for IL21 (rs2055979) and IL21R (rs3093390) gene (P >0.05). P values for genotype and allele frequencies were p=0.188, p=0.769 for IL21 gene, and p=0.144, p=0.179 for IL21R gene respectively.
The mean age of the HCV patients was greater than individuals in healthy controls, also the percentage of men was higher in the HCV group than in the healthy control group; therefore, we used the logistic regression method to omit these confounding variables. After adjustment for covariates including age and gender, in both gene, there is no significant difference between the cases and controls. Results are shown in table 5.

Discussion
In our study, we genotyped two SNPs and evaluated the association of these SNPs with chronic HCV infection. We found no association between IL21 (rs2055979) and IL21R (rs3093390) and susceptibility to chronic HCV infection. To our knowledge, no more extra studies were found about the association of IL21 (rs2055979) and IL21R (rs3093390) with chronic HCV infection in Iranian population. Single nucleotide polymorphisms (SNPs) are the most frequent kinds of genetic variations (26). Many studies supported that SNPs may increase or decrease susceptibility to various diseases (26,27). Cytokines play critical roles in immune responses. On the other hand, cytokine coding genes are polymorphic and some of these polymorphisms may affect the expression of cytokines and also can play roles in the severity and progression of immune-mediated and chronic inflammatory diseases (21). Furthermore, the essential roles of the interleukin receptor genes in function of their exclusive interleukins are not hidden (28,29). IL-21 is a pleiotropic cytokine has been shown to play a significant role in the CD8 T cell response during acute and chronic viral infections. Although the role of IL-21 signaling in the CD4 T cell response to viral infection remains imperfectly defined (30). Recently, there is growing evidence that IL-21 leads to the pathogenesis of chronic inflammatory and autoimmune diseases due to its biological functions (31,32). In addition, the important role for IL-21 as a major helper cytokine during acute HCV infection are proved (11,33). Also lower serum IL-21 levels in chronic HCV patients are detected (34). IL-21 binds to the IL-21 receptor and then can activate some signaling pathways like; Janus kinase (JAK)1, JAK3, STAT1, and STAT3 (14). Recently, discoveries of loss-of function (LOF) mutations in IL-21R in humans have manifested unexpected roles for IL-21 in immune regulation (28). Various single nucleotide polymorphisms within IL-21 and also IL-21R gene are found. Also the association between these polymorphisms and several diseases have been investigated previously. Many studies have investigated associations between genetic variations in these genes and viral infection, but results of these studies have been inconsistent (28,29 (38). The association between genetic variants, defective serum cytokines, improper immune response and susceptibility to various diseases is a topic which has been investigated intensively for hepatitis C and B infection. Recent studies proved that IL-21R polymorphisms are the strongest predictors of sustained virological response to HCV. In addition, IL-21 and IL-21R polymorphisms can be affected on susceptibility for HCV infection in different populations. However, this matter was influenced by viral load and HCV genotype. Up to now, our data and others do not support that IL-21(rs2055979) and IL21R (rs3093390) genetic variants have an association with chronic HCV infection. Since only two SNPs were genotyped, and a small patient sample size was involved in the present study, to confirm this conclusion, further studies with more SNPs and larger patient sample sizes are required.
29. Friedman B, A Hughes. Interleukin 12 receptor β chain (IL12RB2) is a crucial regulatory factor involved in cellmediated immune responses, and genetic variants of the gene encoding IL12RB2 are associated with susceptibility to various immune-related diseases. We previously